Synthesis, functionalization and photo-Bergman chemistry of enediyne bioconjugates

Article abstract of DOI:10.1016/j.bmc.2009.07.059

Methodology is outlined for the chemical synthesis of versatile photo-Bergman enediyne building blocks and their conjugates. Routes to both mono and bis conjugated enediyne templates are detailed together with representative examples of their bioconjugate

Full text of DOI:10.1016/j.bmc.2009.07.059

Bioorganic & Medicinal Chemistry 17 (2009) 6292–6300
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis, functionalization and photo-Bergman chemistry
of enediyne bioconjugates
Paul LaBeaume a, Krista Wager a, Danielle Falcone a, Jane Li a,b, Vladimir Torchilin c, Curtis Castro a,
Coleen Holewa a, Amy E. Kallmerten a, Graham B. Jones a,
*
a Department of Chemistry and Chemical Biology, Northeastern University, Boston, MA 02115, United States
b Albany Molecular Research Laboratories, Albany, NY 01190, United States
c Center for Pharmaceutical Nanomedicine, Northeastern University, Boston, MA 02115, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Methodology is outlined for the chemical synthesis of versatile photo-Bergman enediyne building blocks
and their conjugates. Routes to both mono and bis conjugated enediyne templates are detailed together
with representative examples of their bioconjugates, nanoconjugates, PEG derivatives and water soluble
salts. The immunocompetence of antibody conjugates is retained, and application in the form of reagents
for photodynamic therapy (PDT) advanced.
Received 2 June 2009
Revised 17 July 2009
Accepted 21 July 2009
Available online 26 July 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Enediyne
Photo-Bergman
Bioconjugate
Antibody conjugate
Nanoparticles
PDT
1. Introduction
efficient routes to substituted photoactivated enediyne templates,
and investigating their bioconjugation chemistries. In pursuit of
Isolated from soil bacteria, enediynes are among the most po-
tent groups of antineoplastic agents ever discovered.1 Introduced
in 2000, MylotargÒ an anti-CD33 antibody conjugate of the ened-
iyne calicheamicin, was the first ever Mab-cytotoxin conjugate to
be approved by the FDA, and is clinically effective against acute
myeloid leukemia (AML).2 Through complex triggering mecha-
nisms the natural enediyne pharmacophores are converted to cyto-
toxic cyclic diradical species via a process commonly referred to as
the Bergman cycloaromatization, ultimately abstracting hydrogen
atoms from surrounding macromolecules leading to DNA damage
and cell death.3 A number of designed enediyne prodrugs have
been developed, with a variety of inbuilt triggering mechanisms.
Perhaps the most simplistic of all relies on the photo-Bergman
reaction, wherein otherwise shelf-stable enediyne substrates are
transformed to cytotoxic diradicals on demand through irradiation.
The first and perhaps best studied version of this transformation is
exemplified by bis-phenyl enediyne 1 which transforms to diyl 2
under UV irradiation, en route to aryl 3 (Scheme 1).3 The basic
photo-Bergman pharmacophore however has limited application
in drug design due to the lipophilic nature of the substrates.
Accordingly, we and others have become interested in developing
these goals, as documented herein, we have concentrated on (i)
preparation of versatile enediyne building blocks (ii) derivatization
to form water soluble analogues (iii) coupling to form PEG-ylated
derivatives (iv) formation of antibody conjugated chimeras and
(v) investigation of the potential to form nanoparticle coupled
derivatives.
O
hP67.6
Mab
O
N
H
O
N
N
CH3
SS
H
O
NHCO2CH3
OH
CH3
S
CH3
O
HO
O
O
O
O
O
NH
OH
OCH3
OCH3
H3C
OCH3
NHC2H5
O
I
O
MylotargR
O
H3C
HO
* Corresponding author. Tel.: +1 617 373 8619; fax: +1 617 373 8795.
E-mail address: gr.jones@neu.edu (G.B. Jones).
H3CO
OH
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.07.059

P. LaBeaume et al. / Bioorg. Med. Chem. 17 (2009) 6292–6300
6293
H
H
Ph
Ph
hν
2H
Ph
Ph
Ph
Ph
1
2
3
Scheme 1. The basic photo-Bergman cycloaromatization.
1.1. Preparation of building blocks
Though a variety of amines were screened, the corresponding
ammonium salt(s) from DABCO proved the most effective with
products 13 and 14 isolated in good yield and essentially stable
as crystalline salts (Scheme 3). The salts proved to be freely sol-
uble in polar organic solvents and buffering media, and more
importantly also in water.
A variety of efficient Pd-mediated routes to differentially substi-
tuted aryl enediynes have been developed, and we elected to com-
mence from di-iodobenzene 4, which can be transformed easily
into enediyne template 5 (Scheme 2). Conversion to phenacetic es-
ter 6 allowed formation of either acid 7 or ethanol 8 as needed in
excellent yield. Conditions were also developed for direct coupling
to form enediyne alcohol 9, which in turn allowed us to access
enediyne thiol 10. In order to expand the repertoire of the photo-
Bergman reaction and fully explore bioconjugation strategies we
also executed a route to bis-benzyl alcohol 12. This merely neces-
sitated formation of parent unsubstituted enediyne 11 followed by
double coupling–deprotection to give the desired product in good
yield.
1.3. Formation of PEG-ylated derivatives
With a variety of mono and bis-functionalized enediyne tem-
plates in hand we began to investigate formation of PEG-ylated
derivatives. PEG-ylation is now a mainstream strategy in drug
development, enhancing the properties of lipophilic molecules
and in the case of targeting of solid tumours, providing enhanced
circulatory and localizing benefit via the RES mechanism.4 Conver-
sion of 9 into the PEG-ylated ester 17 was effective using a PEG dia-
cid under EDCI coupling (Scheme 4). Attempts to effect the same
coupling on diol 12 met with difficulty, invariably leading to insep-
arable mixtures of mono and bis esters 18 and 19. Remedy was
found by conversion to the bis succinate 20, allowing efficient pro-
duction of the bis-PEG-ylated ester 21 through carbodiimide
coupling.
1.2. Water soluble analogues
Though carboxylate 7 and diol 12 have some solubility in
polar solvents, we wished to also advance formation of more
versatile ammonium salts, and investigated transformation of
both 9 and 12 via their corresponding mesylate derivatives.
Ph
H
1. Pd(PPH3)2Cl2, CuI,
n-BuNH2 (59%)
Ph
I
I
2. Pd(PPh3)Cl2
CuI, Et3N (57%)
TMS
4
5
3. TBAF, THF (100%)
1. Pd(PPh3)2, CuI,
n-BuNH2 (67%)
Pd(PPh3)2Cl2
CuI,Et3N
p-I-C6H4CH2OH
Pd(PPh3)2Cl2,
CuI, Et3N
p-I-C6H4CH2CO2Me
2. K2CO3, MeOH (99%)
(99%)
(81%)
Ph
H
Ph
11
H
CH2CO2Me
6
OH
1. Pd(PPh3)2, CuI,
Et3N (53%)
OTBS
9
2 eq Dibal-H
99%
LiOH, THF/H2O
(90%)
1. PPh3, (NCO2i-Pr)2
CH3COSH (60%)
I
2. TBAF, THF
(99%)
2. LiAlH4 (82%)
OH
Ph
Ph
Ph
CH2CO2H
12
CH2CH2OH
OH
7
8
10
Scheme 2. Synthesis of photoenediyne building blocks.
CH2SH

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P. LaBeaume et al. / Bioorg. Med. Chem. 17 (2009) 6292–6300
OH
OSO2CH3
OSHO2CH3
N
N
N
N
1. MesCl
2. R1R2R3N
N
N
9 R= H
12 R= CH2OH
14
13
R
OSO2CH3
OSO2CH3
OSO2CH3
N
N
Ph
Ph
Ph
15
N
16
OSO2CH3
Scheme 3. Improved water solubility through benzylammonium enediyne salts.
1.4. Antibody conjugates
coupling of enediyne 10 with HAuCl4 gave nanoconjugate 27
(Scheme 7).11 The morphology of the Au derivatives was assessed
through TEM analysis (Fig. 1). The nanoparticles showed an aver-
age core size of approx. 5.0 nm, anticipated based on a benzyl mer-
capto Au nanoparticle prepared as a standard. TEM images were
re-taken after a period of one month to verify the shelf-stability
of the nanoparticles. NMR proved to be an excellent means to as-
sess conversion, with conjugated and non-conjugated versions
showing characteristic spectra.
In summary, readily available linear (aryl) enediynes can be
prepared and efficiently functionalized allowing conversion to a
variety of thermally stable derivatives. The inherent hydrophobic-
ity of the enediyne cores can be addressed through PEG-ylation
and derivatization as ammonium salts. In all cases the enediynes
undergo photo-induced cycloaromatization to yield arene prod-
ucts. Bioconjugation in the form of monoclonal antibody deriva-
tives is achieved either through amide or ester linked PEG’s and
immunocompetence is retained. Finally, Au nanoparticle enediyne
conjugates can be prepared via a two-phase method. The method-
ology described herein is flexible and efficient, and we expect
application in bioorganic, medicinal and materials photochemistry.
The DNA cleavage capacity of these photoactivated systems has
been confirmed, lending the possibility for the design of targeted
applications in a number of areas.
With PEG ester chemistry established, we turned our attention
to preparation of monoclonal antibody conjugates. Direct coupling
of PEG carboxylate 17 with the S-NHS ester of an available cardiac
specific antibody (2G4) gave derivative 22 (Scheme 5).5 Alterna-
tively, coupling of template 7 via linker 23 followed by reductive
amination, allowed preparation of congener 24.6 In both cases
immunochemical analysis confirmed activity of the derivatized
products by ELISA.7
1.5. Photo-Bergman chemistry
The enediynes, whether in parent, ammonium salt, PEG-ylated,
or bioconjugated form underwent photo-Bergman cyclization to
give the corresponding product arenes 26 via presumed diyl inter-
mediates 25 (Scheme 6). Optimal conditions involve irradiation
through a low pressure Hg lamp (450 W) using isopropanol as
hydrogen atom donor. Conversion to the cycloaromatized species
is conveniently monitored utilizing UV/vis spectroscopy based on
the characteristic increase in wavelength associated with aromati-
zation. NMR and MS confirmed the identity of products, in the case
of PEG-ylated derivatives and bioconjugates using MALDI-MS.
To assess DNA cleaving ability of the photo-Bergman substrates,
irradiation of 17 in the presence of bacteriophage (UX174 RfI) DNA
was conducted. Correcting for control photodegradation, 17 largely
induced concentration dependent conversion to type II DNA (24%),
with the onset of minimal conversion to type III based on random
single-stranded cutting events. This moderate level of degradation
presumably reflects affinity of the enediyne warhead itself for DNA
as has been observed in other cases,8 however targeting entities
are known to improve affinity.9 The study confirms however that
in PEG-ylated form, the enediynes are still able to effectively form
the cytotoxic diyl radicals, supporting the design approach.
2. Experimental procedures
NMR spectra were obtained either on a Varian Mercury 300
(300 MHz) or a Varian Inova 500 (500 MHz) spectrometer. Mass
spectra were obtained on a Hewlett Packard 6890 Plus GC with
5973 Mass Selective Detector and Agilent HP Ultra 1,
25 m ꢀ 0.2 mm column with a 0.11
lm film thickness. The elec-
tron impact ionization source was run at 70 eV. MALDI was con-
ducted on an Applied Biosystems 4800 MALDI-TOF/TOF. High
resolution TEM analyses were performed on JEOL, JEM 1010 and
the images captured using an Ex41B 4MP bottom mount CCD cam-
era system (Advanced Microscopy Techniques). Thin layer chroma-
tography (TLC) was performed using Silica Gel 60 F524 precoated
plates (Scientific Adsorbants Inc.) Preparative thin layer chroma-
tography was carried with Silica Gel GF (Analtech, Inc.). Flash chro-
1.6. Future applications—Nanoparticle conjugates
Functionalized Au nanoparticle derivatives are becoming sta-
ples of investigational medicinal chemistry,10 and we sought to
demonstrate coupling of an enediyne building block. Reductive

P. LaBeaume et al. / Bioorg. Med. Chem. 17 (2009) 6292–6300
6295
Ph
PEG diacid
(32%)
9
EDCI, DMAP, CH2Cl2
O
O
O
O
OH
17
12
O
12
HOOC
O
O
COOH
O
n
EDCI, Et3N
O
O
O
O
<10%
O
COOH
O
O
COOH
8
n
O
O
O
O
O
COOH
OH
O
8
19
18
O
99%
O
O
O
O
O
O
COOH
8
O
OH
HO
O
O
OH
n
EDCI, Et3N (34%)
O
O
O
O
O
O
COOH
8
21
20
O
OH
Scheme 4. Optimized route to PEG-ylated photo-enediynes.
matography was performed using Silica Gel 60 (230–400 mesh,
Whatman Inc.). All reactions were carried out under anhydrous
conditions, under inert atmosphere (nitrogen or argon) with dry,
freshly distilled solvents and flame dried glassware unless other-
wise noted. All PEG reagents were purchased from Laysan Bio, Inc.
2.1. 1-Iodo-2-(ethynylphenyl)benzene
To a solution of 1,2-diiodobenzene (4) (10 g, 30.03 mmol) in
diethylether (30 mL) was added dichloro(bistriphenylphos-
phine)palladium (0.85 g, 1.21 mmol). The solution was degassed
Ph
EDC, S-NHS
17
2G4 mAb
(70%)
O
7
22
O
2G4
O
O
N
H
12
O
1. DCC, CH2Cl2 (96%)
Ph
O
H2N
O
n
MW=400
MeO
OMe
O
24
O
2G4
23
N
H
O
N
2. Amberlyst-15
CH3COCH3, H2O (90%)
H
6
NaCNBH3, HEPES
pH 7.4 2G4 mAb
3.
Scheme 5. Antibody conjugation of the photo-enediynes.

6296
P. LaBeaume et al. / Bioorg. Med. Chem. 17 (2009) 6292–6300
Ph
Ph
Ph
hv 450W 3 hr
isopropanol
R
R
26
R
25
R = OH (9)
= O-PEG (17)
= O-PEG-2G4 (22)
= COOH (7)
=
NC8H11 OSO2CH3 (16)
Scheme 6. Photo-Bergman cyclization of enediynes.
(N2) for 45 min. In a second flask copper iodide (0.49 g, 2.55 mmol)
and n-butyl amine (1.78 g, 24.3 mmol) were added to a degassed
solution of phenyl acetylene (0.62 g, 6.0 mmol) in ether (20 mL).
The diiodobenzene solution was cannulated into the alkyne solu-
tion and the resulting mixture stirred for 12 h at 25 °C and then
saturated ammonium chloride (20 mL) added. Ethyl acetate
(20 mL) was added to the biphasic mixture, and the organic layer
washed with water (20 mL) and the combined aqueous layers were
extracted with ethyl acetate (2 ꢀ 30 mL). The combined organic
layers were washed with brine (30 mL), dried (MgSO4) and concen-
trated in vacuo. The crude solid was subjected to flash chromatog-
raphy (hexanes) to yield the title compound (1.08 g, 59%) as a
white solid (mp 62–65 °C); Rf 0.36 (hexanes); 1H NMR (300 MHz,
CDCl3): 7.87 (dd, 1H, J = 3.9, 6.3 Hz), 7.61 (m, 2H), 7.5 (dd, 1H,
J = 4.5, 7.2 Hz), 7.35 (m, 4H), 7.03 (dd, 1H, J = 3.6, 6.0 Hz); 13C
NMR (75 MHz, CDCl3): 138.7, 132.7, 132.6, 129.5, 129.3, 128.7,
128.5, 127.8, 122.0, 101.2, 93.0, 91.6; HRMS (CI), m/z C14H9I
(M+H)+: calcd 304.9828, obsd 304.9828.
2.2. 1-(Trimethylsilylethynyl)-2-(ethynylphenyl)benzene
Figure 1. TEM image of Au enediynenanoparticles 27.
To a degassed solution of 1-iodo-2-(ethynylphenyl)benzene
(1.08 g, 3.55 mmol) in triethylamine (10 mL) was added
dichloro(bistriphenylphosphine)palladium (0.1 g, 0.14 mmol) and
copper iodide (0.055 g, 0.29 mmol). The solution was degassed and
trimethylsilylacetylene (0.46 g, 4.67 mmol) added. The solution
was again degassed and then stirred for 12 h at 25 °C. The solution
was quenched with saturated ammonium chloride (10 mL). Ethyl
acetate (20 mL) was added to the biphasic mixture. The organic layer
was washed with water (20 mL) and the combined aqueous layers
were extractedwithethylacetate (2 ꢀ 30 mL). Thecombinedorganic
layers were washed with brine (30 mL), dried (MgSO4) and concen-
trated in vacuo. The reside was subjected to column chromatography
(1:99, ethylacetate/hexanes) to yield the title compound as a yellow
oil (0.55 g, 57%); Rf: 0.25 (hexanes); 1H NMR (300 MHz, CDCl3): 7.56
(m, 2H), 7.50 (m, 2H), 7.32 (m, 3H), 7.23 (m, 2H), 0.26 (9H); 13C NMR
(75 MHz, CDCl3): 132.5 131.9, 128.6, 128.5, 128, 0.0; HRMS (CI), m/z
C19H18Si (M+H)+: calcd 275.1256, obsd 275.1256.
to 0 °C. Tetrabutylammonium fluoride (2.79 g, 10.7 mmol, 1.0 M
in THF) was added dropwise. The reaction mixture was stirred
for 2.5 h at 0 °C. Water (15 mL) was added slowly to quench the
reaction. The biphasic mixture was extracted with ethyl acetate
(2 ꢀ 30 mL). The combined organic layers were washed with brine
(30 mL), dried (MgSO4) and the solution filtered through a short
plug of silica gel and concentrated in vacuo to yield the title com-
pound as a yellow oil (0.53 g, 100%). Rf: 0.27 (hexanes); 1H NMR
(300 MHz, CDCl3): 7.59–7.52 (m, 4H), 7.36–7.25 (m, 5H), 3.36 (s,
1H); 13C NMR (75 MHz, CDCl3): 132.8, 132.0, 128.6, 128.1, 127.9,
126.6, 124.9, 123.4, 93.78, 88.1, 88.3; HRMS (CI), m/z C16H10
(M+H)+: calcd 203.0863, obsd 203.0861.
2.4. (4-Iodo-phenyl)-acetic acid methyl ester
A 25 mL round bottom flask was charged with a mixture of fluo-
roboric acid (1.0 mmol, 310 lL of 48% in H2O) and 4-iodophenyl-
2.3. 1-(Ethynylphenyl)-2-ethynylbezene (5)
acetic acid (0.262 g, 1.0 mmol) in dichloromethane (15 mL). The
mixture was cooled to 0 °C and stirred vigorously while trimethyl-
silyldiazomethane (0.56 mL, 1.8 M in hexanes, 1.0 mmol) was
added dropwise over 5 min. With continued vigorous stirring at
A solution of 1-(trimethylsilylethynyl)-2-(ethynylphenyl)ben-
zene (0.73 g, 2.66 mmol) in tetrahydrofuran (20 mL) was cooled
Ph
1. PPh3(+NCO2i-Pr)2
CH3COSH
2. LiAlH4
HAuCl4 3H2O
NaBH4
9
10
27
S
Au
Scheme 7. Synthesis of Au nanoparticles stabilized by thiolated enediyne.

P. LaBeaume et al. / Bioorg. Med. Chem. 17 (2009) 6292–6300
6297
0 °C, three additional portions of trimethylsilyldiazomethane
(0.28 mL, 0.5 mmol), (0.14 mL, 0.25 mmol) and (0.14 mL,
0.25 mmol) were added dropwise at intervals of 20 min then mix-
ture was stirred at 0 °C for an additional 30 min. Water (30 mL)
was added slowly, then the mixture was extracted with dichloro-
methane (10 mL), the organic layer washed with water (10 mL),
brine (10 mL), then dried (MgSO4) and concentrated in vacuo.
The crude residue was purified by flash chromatography (ethyl
acetate/hexanes, 15:85), to yield the title compound as a pale yel-
low oil (0.252 mg, 91%). 1H NMR (300 MHz, CDCl3): 7.68 (d,
J = 8.7 Hz, 2H), 7.06 (d, J = 8.7 Hz, 2H), 3.72 (s, 3H), 3.59 (s, 2H);
13C NMR (75 MHz, CDCl3): 171.6, 137.9, 133.8, 131.5, 92.9, 52.4,
40.8; HRMS (EI), m/z C9H9IO2 (M+H)+: calcd 276.9725, obsd
276.0001.
flash chromatography (20:80, ethyl acetate/hexanes) to afford the
title compound (0.57 g, 99%) as
colourless oil. 1H NMR
a
(300 MHz, CDCl3): 7.59–7.12 (m, 13H) 3.88 (t, J = 6.0 Hz, 2H), 2.9
(t, J = 6.0 Hz, 2H), 2.1 (br, s, OH); 13C NMR (75 MHz, CDCl3):
139.4, 132.1, 131.9, 129.3, 128.6, 127.8, 126.1, 123.5, 121.7, 93.7,
88.5, 88.4, 63.6, 39.3; HRMS (ESI), m/z C24H18O (M+H)+: calcd
323.1435, obsd 322.2074.
2.8. 4-(2-Phenylethynyl-phenylethynyl)-benzyl alcohol (9)
To a degassed solution of 1-(ethynylphenyl)-2-ethynylbenzene
(0.53 g, 2.62 mmol) in triethylamine (35 mL) was added 4-iodob-
enzyl alcohol (0.737 g, 3.15 mmol), dichloro(bis triphenylphos-
phine)palladium (0.074 g, 0.11 mmol) and copper iodide (0.04 g,
0.21 mmol). The solution was degassed again (45 min N2 purge)
and then stirred at room temperature for 4 h. Saturated ammo-
nium chloride (30 mL) was added followed by ethyl acetate
(30 mL). The organic layer was washed with water (30 mL) and
the combined aqueous layers extracted with ethyl acetate
(2 ꢀ 40 mL). The combined organic layers were washed with brine,
dried (MgSO4) and concentrated in vacuo. The residue was purified
by flash chromatography (70:30, hexanes/ethyl acetate) to yield
the title compound (0.65 g, 81%) as a tan solid. (mp 95–97 °C);
1H NMR (300 MHz, CDCl3): 7.59–7.25 (m, 13H), 4.71 (s, 2H), 1.75
(br s, 1H); 13C NMR (75 MHz, CDCl3): 141.4, 132.1, 132.0, 131.9,
128.7, 128.6, 128.3, 127.1, 126.1, 123.5, 122.8, 93.8, 93.7, 88.6,
88.5, 65.2; HRMS (CI), m/z C23H16O (M+H)+: calcd 309.1281, obsd
309.1280.
2.5. 4-(2-Phenylethynyl-phenylethynyl)-phenyl acetic acid
methyl ester (6)
To a degassed solution of 1-(ethynylphenyl)-2-ethynylbenzene
(0.28 g, 1.4 mmol) in triethylamine (20 mL) was added (4-iodo-
phenyl)acetic acid methyl ester (0.46 g, 1.68 mmol), dichloro-
(bistriphenylphosphine)palladium (0.04 g, 0.05 mmol), and copper
iodide (0.02 g, 0.12 mmol). The solution was degassed again
(45 min N2 purge) and then stirred at 25 °C for 5 h. Saturated
ammonium chloride (20 mL) was added, followed by dichloro-
methane (20 mL). The organic layer was washed with water
(20 mL) and the combined aqueous layers were extracted with
dichloromethane (20 mL). The combined organic layers were
washed with brine (20 mL), dried (MgSO4) and concentrated in va-
cuo. The residue was purified by flash chromatography (85:15,
hexanes/ethyl acetate) to yield the title compound as a yellow oil
(0.44 g, 90%). 1H NMR (300 MHz, CDCl3): 7.57–7.25 (m, 13H),
3.71 (s, 3H), 3.64 (s, 2H); 13C NMR (75 MHz, CDCl3): 171.8, 134.5,
132.4, 132.1, 131.9, 129.6, 128.7, 128.6, 128.2, 127.7, 126.1,
123.5, 122.4, 93.8, 93.6, 88.7, 88.5, 52.4, 41.3; HRMS (EI), m/z
(M+H)+: C25H18O2 calcd 351.1385, obsd 350.1099.
2.9. 4-(2-Phenylethynyl-phenylethynyl)-benzyl thioacetate
Diisopropylazodicarboxylate (0.21 mL, 1.06 mmol) was added
dropwise to a solution of triphenylphospine (0.26 g, 1.05 mmol)
in THF (2.5 mL) at 0 °C. The solution was stirred for 30 min at
0 °C then
a solution of (4-((2-phenylethynyl) phenyl)ethy-
nyl)phenyl)methanol (0.16 g, 0.05 mmol) and thiolacetic acid
(0.08 g, 1.05 mmol) in THF (2 mL) added dropwise. The mixture
was stirred for 1 h at 0 °C then warmed to 25 °C and stirred for
an additional 1 h. The solution was extracted with dichlorometh-
ane (10 mL) and the organic layer washed with saturated sodium
2.6. 2-(4-((2-(Phenyethynyl)phenyl)ethynyl)phenyl)acetic acid
(7)
Lithium hydroxide monohydrate (0.97 g, 2.48 mmol) was added
to a solution of 4-(2-phenylethynyl-phenylethynyl)-phenyl acetic
acid methyl ester (0.087 g, 0.248 mmol) in THF/water 5:4
(13 mL). The resulting mixture was stirred for 24 h at 25 °C then
the solution was condensed in vacuo. The reside was diluted with
dichloromethane (5 mL), washed with water (10 mL), brine
(10 mL), dried (MgSO4) and concentrated in vacuo. The residue
was purified by flash chromatography (20:80, ethyl acetate/hex-
anes) to afford the title compound as a colourless oil (0.075 g,
90%). 1H NMR (300 MHz, CDCl3): 7.62–7.45 (m, 6H), 7.40–7.20
(m, 7H), 3.68 (s, 2H); 13C NMR (75 MHz, CDCl3): 150.7, 132.7,
132.1, 132.0, 131.9, 129.7, 128.7, 128.6, 128.2, 126.1, 126.0,
123.5, 122.7, 93.8, 93.4, 88.8, 88.5, 41.2; HRMS (ESI), m/z
C24H16O2 (M+H)+: calcd 337.1299, obsd 337.2001.
bicarbonate (4 ꢀ 2 mL). The solution was concentrated in vacuo
1
to approx.
original volume then diluted with an equal volume
4
of a 1:1 hexanes/ether mixture. The solution was filtered through
a pad of Celite and dried (MgSO4). The solution was again, diluted
with an equal volume of hexanes/ether and then filtered over a pad
of silica gel and concentrated in vacuo. The reside was subjected to
flash chromatography (100% dichloromethane) to yield the title
compound as a yellow oil (0.11 g, 58%). 1H NMR (300 MHz, CDCl3):
7.58–7.25 (m, 13H), 4.12 (s, 2H), 2.36 (s, 3H); 13C NMR (75 MHz,
CDCl3): 194.9, 138.1, 132.3, 131.9, 131.6, 128.9, 128.4, 128.0,
125.8, 123.3, 122.3, 93.6, 93.3, 88.6, 88.2, 33.3, 30.3. HRMS (CI),
m/z C25H18OS (M+H)+: calcd 367.1156, obsd 367.0902.
2.10. 4-(2-Phenylethynyl-phenylethynyl)-benzyl thiol (10)
2.7. 2-(4-((2-(Phenylethynyl)phenyl)ethynyl)phenyl)ethanol (8)
A solution of lithium aluminium hydride (0.013 g, 0.34 mmol,
1 M) in ether (0.6 mL) was added dropwise to a solution of 4-(2-
phenylethynyl-phenylethynyl)-benzyl thioacetate (0.11 g, 0.3 mmol)
in ether (1.5 mL) at 0 °C. The mixture was stirred at 0 °C for 20 min,
then warmed to 25 °C and stirred for an additional 30 min. The mix-
ture was cooled to 0 °C and treated with hydrochloric acid (3 N,
ꢂ1.5 mL) until all solid dissolved. The solution was diluted sequen-
tially with ether (2 mL), water (0.2 mL), sodium hydroxide (0.02
mL, 15% in water) then water (0.006 mL). The mixture was warmed
to room temperature, stirred for 15 min then extracted with ether
(2 ꢀ 2 mL). The combined organic layers were washed with brine
Diisobutyl aluminium hydride (DIBAL-H) in toluene (1 M,
1.778 mmol, 3 mL) was added to a solution of 4-(2-phenyleth-
ynyl-phenylethynyl)-phenyl acetic acid methyl ester (0.623 g,
1.778 mmol) in THF (13 mL) at ꢁ78 °C. The mixture was stirred
for 30 min then an additional equivalent of DIBAL-H added. Meth-
anol (5 mL), then water (0.5 mL) was added, then the solution was
filtered. The filtrate was extracted with dichloromethane
(3 ꢀ 5 mL), washed with water (10 mL), brine (10 mL), dried
(MgSO4) and concentrated in vacuo. The residue was purified by

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P. LaBeaume et al. / Bioorg. Med. Chem. 17 (2009) 6292–6300
(5 mL), dried (MgSO4) and concentrated in vacuo to yield the title
compound as a yellow oil (0.8 g, 82%). 1H NMR (300 MHz, CDCl3):
7.57–7.19 (m, 13H), 3.75 (d, 2H, J = 7.8 Hz), 1.77 (t, SH J = 6.0 Hz);
13C NMR (75 MHz, CDCl3): 146.8, 138.1, 132.2, 131.9, 128.7, 128.2,
127.3, 125.4, 123.5, 122.3, 93.6, 93.2, 88.6, 88.4, 29.9. HRMS (CI),
m/z C23H16S (M+H)+: calcd 324.10, obsd 324.20.
lyloxy)methyl)phenyl)ethynyl)benzene in tetrahydrofuran at 0 °C
(Ar atmosphere) and stirred for 30 min. Water (20 mL) was added.
The solution was condensed in vacuo, diluted with ethyl acetate
(300 mL) and the organic layer washed with water (200 mL) and
brine (100 mL), dried (MgSO4) and condensed in vacuo. The residue
was purified by flash chromatography (2:1 ethyl acetate/hexanes)
to afford the title compound (0.133 g, 98%) as a white solid.
Mp = 146–147 °C; TLC (hexanes/ethyl acetate = 1:1): Rf 0.18; 1H
NMR (500 MHz, CDCl3): d 7.54–7.59 (m, 6H), 7.34–7.38 (m, 4H),
7.30–7.33 (m, 2H), 4.73 (s, 4H), 1.4–1.9 (br s, 2H); 13C NMR
2.11. 1,2-Bis((trimethylsilyl)ethynyl)benzene
Pd(PPh)2Cl2. (0.032 g, 3 mol %) and CuI (0.014 g, 5 mol %) were
added to a degassed (Ar 30 min) solution of 1,2-diiodobenzene
(75 MHz, CDCl3):
d 141.32, 131.97, 131.91, 128.19, 127.00,
125.93, 122.66, 93.57, 88.52, 65.11; HRMS C24H18O2 (CI), m/z
(0.500 g, 198
lL, 1.516 mmol) in triethylamine (5 mL). The solution
(M)+: calcd 338.1307, obsd 338.1307.
was degassed (Ar, 30 min) then TMS–acetylene (535
lL, 2.5 equiv)
added and the mixture stirred for 16 h at 25 °C. Saturated aqueous
NH4Cl (5 mL) was added followed by ethyl acetate (10 mL). The
layers were separated and the organic layer was washed with
water (2 ꢀ 10 mL). The aqueous layers were back extracted with
ethyl acetate (2 ꢀ 10 mL) and the combined organic extracts
washed with brine (10 mL), dried over MgSO4 and concentrated
in vacuo. The residue was purified by flash column chromatogra-
phy to afford the title compound (0.272 g, 66.4%) as a yellow
oil.12 TLC (hexanes/ethyl acetate = 9:1): Rf 0.53; 1H NMR
(300 MHz, CDCl3): d 7.45 (dd, J = 6.3 Hz, 3.3 Hz, 2H), 7.19–7.23
(m, 2H), 0.27 (s, 18H); 13C NMR (75 MHz, CDCl3): d 132.24,
127.99, 125.75, 103.23, 98.36, 0.00.
2.15. Standard procedure for photo-cyclization of enediynes
A solution of enediyne (5 mg, 0.005% in HPLC grade isopropa-
nol) is added to a 14 mL quartz UV cuvette and degassed (Ar,
30 min). In a photochemical box, a 0.01 M potassium chromate
solution filter is placed between the lamp and the solution, and
irradiation initiated using a 450 W Hanovia low-pressure Hg lamp.
The reaction is stirred for up to 3 h (N2 atmosphere) and monitored
by both UV–vis and mass spectrometry to determine the rate and
extent of formation of cylcloaromatized product. Representative
kmax 16 = 275 nm (26 = 305 nm); 22 = 308 nm (26 = 364 nm). Fluo-
rescence quantum yields based on analysis of 9/26 were consistent
with those reported for 1.15
2.12. 1,2-Diethynylbenzene (11)
Potassium carbonate (0.542 g) was added to a solution of 1,2-
bis((trimethylsilyl) ethynyl)benzene (0.212 g, 0.784 mmol) in
methanol/THF (1:1, 80 mL). The resulting solution was stirred at
25 °C (Ar atmosphere) for 30 min then diluted with ether
(300 mL). The mixture was washed with saturated ammonium
chloride (4 ꢀ 150 mL), water (4 ꢀ 150 mL) then dried (MgSO4)
and condensed in vacuo to afford the title compound (0.098 g,
99%) as a clear oil.13 TLC (hexanes/ethyl acetate = 9:1): Rf 0.42;
1H NMR (300 MHz, CDCl3): d 7.50 (dd, J = 6.3 Hz, 2H), 7.24–7.31
(m, 2H), 3.33 (s, 2H); 13C NMR (75 MHz, CDCl3): d 132.87, 128.76,
125.30, 82.09, 81.49.
2.16. Photoinduced DNA cleavage
U
X174 (RfI) DNA was irradiated (Hanovia 450 W medium pres-
sure Hg lamp) in TRIS–HCl at pH 8.5 with DNA (50 M in base
l
pairs) as control or with 17 at 10ꢁ6 M for 3 h at 37 °C. Crude mix-
tures were subjected to gel electrophoresis (10% agarose), stained
with ethidium bromide, then subjected to scanning densitometry.
Under these conditions up to 20–25% of RfII DNA is produced (cor-
rected for 3–5% produced on photoirradiation in absence of sub-
strate) together with 2–5% RfIII form, derived from random
degradation of RfII form. Irradiation in the presence of cycloaroma-
tized substrates or incubation in the presence of enediynes re-
sulted in no increase in DNA degradation over controls.
2.13. 1,2-Bis((4-((tert-butyldimethylsilyloxy)methyl)phenyl)-
ethynyl)benzene
2.17. 1-(4-((2-(Phenylethynyl)phenyl)ethynyl)benzyl)-4-aza-1-
azoniabicyclo[2.2.2]octane methanesulfonate (salt) (13)
Pd(PPh)2Cl2. (0.0445 g, 8 mol %) and CuI (0.1208 g, 8 mol %) were
added to a degassed (Ar, 30 min) solution of 1,2-diethynylbenzene
(0.100 g, 0.793 mmol) and 4-((tertbutyldimethylsilyloxy)methyl)-
phenyl iodide14 (0.607 g, 1.75 mmol) in triethylamine (25 mL). The
resulting solution was stirred at 25 °C (Ar atmosphere) for 5 h then
diluted with saturated ammonium chloride (5 mL) and ethyl acetate
(300 mL). The organic layer was washed with brine (100 mL), dried
(MgSO4) and condensed in vacuo. The residue was purified by flash
column chromatography (19:1 hexanes/ethyl acetate) to afford the
title compound (0.240 g, 53%) as a yellow solid. Mp = 109–111 °C;
TLC (hexanes/ethyl acetate = 19:1): Rf 0.35; 1H NMR (300 MHz,
CDCl3): d 7.48–7.60 (m, 4H), 7.21–7.36 (m, 8H), 4.76 (s, 4H), 0.95
(s, 18H), 0.11 (s, 12H); 13C NMR (75 MHz, CDCl3): d 155.66, 147.16,
136.96, 136.83, 133.12, 131.21, 131.16, 127.04, 99.00, 93.30, 69.93,
31.20, 23.63, 0.0; HRMS (CI), m/z C36H46O2Si2 (M+H)+ calcd
567.3116, obsd 567.3115.
Triethylamine (41
lL, 3 equiv) and methanesulfonyl chloride
(15 L, 2 equiv) was added to a solution of 4-(2-phenylethynyl-
l
phenylethynyl)-benzyl alcohol in dichloromethane (4 mL) at 0 °C
(Ar atmosphere) then stirred at 25 °C for 1 h. The resulting solu-
tion was diluted with ethyl acetate (25 mL) and washed with cit-
ric acid (5%, 30 mL), sodium bicarbonate (5%, 30 mL) and brine
(30 mL), dried (MgSO4) and condensed in vacuo. This residue
was added dropwise to a solution of 1,4-diazabicyclo[2.2.2]octane
(DABCO) (0.0086 g, 0.7666 mmol) in acetonitrile (2 mL) at 0 °C.
The resulting solution was stirred at 0 °C for 30 min, warmed to
25 °C and condensed in vacuo. The residue was recrystallized
from hexanes, methanol and ethyl acetate to afford the title com-
pound 0.036 g, 94%) as a yellow solid. Mp 60–61 °C; 1H NMR
(500 MHz, CDCl3): 7.51–7.57 (m, 8H), 7.34–7.39 (m, 2H), 7.24–
7.33 (m, 3H), 4.89 (s, 2H), d 3.59 (t, J = 7.5 Hz, 6H), 3.13 (t,
J = 7.5 Hz, 6H), 2.84 (s, 3H); 13C NMR (75 MHz, CD3OD):
d
2.14. (4,40-(1,2-Phenylenebis(ethyne-2,1-diyl))bis(4,1-phenylene))-
dimethanol (12)
135.54, 134.20, 134.10, 134.02, 133.53, 130.69, 128.94, 128.01,
127.94, 127.14, 125.24, 95.39, 93.90, 92.08, 89.65, 69.60, 54.40,
46.98, 40.28; HRMS C29H27N2 (EI), m/z (M)+: calcd 403.2169,
þ
Tetrabutylammonium fluoride (815
lL, 0.815 mmol, 1 M in
THF) was added to a solution of 1,2-bis((4-((tert-butyldimethylsi-
obsd 403.2174.

P. LaBeaume et al. / Bioorg. Med. Chem. 17 (2009) 6292–6300
6299
2.18. 1,10-(4,40-(1,2-Phenylenebis(ethyne-2,1-diyl))bis(4,1-
phenylene))bis(methylene)bis(4-aza-1-
azoniabicyclo[2.2.2]octane) methanesulfonate (14)
in vacuo and diluted with 49:1 hexanes/methanol (10 mL). After
vigorous stirring the methanol layer was removed, the hexanes
layer was warmed to 25 °C and methanol (1 mL) added. This pro-
cess was repeated three times and the methanol extracts were
combined, dried (MgSO4), and condensed in vacuo to afford the ti-
tle compound (0.008 g, 52%) as a white solid. Mp 55–57 °C; 1H
NMR (500 MHz, CD3OD): d 7.41–7.77 (m, 22H), 4.66 (s, 4H), 4.64
(s, 4H), 2.98 (s, 12H), 2.72 (s, 6H); 13C NMR (125 MHz, CD3OD): d
134.82, 134.50, 133.40, 132.20, 132.09, 130.58, 130.19, 129.27,
128.88, 127.22, 126.55, 93.35, 91.20, 70.02, 69.42, 43.09, 39.67;
Triethylamine (22.2
lL, 6 equiv) and methanesulfonyl chloride
(12.42
l
L, 6 equiv) was added to a solution of (4,40-(1,2-phenyl-
enebis(ethyne-2,1-diyl))bis(4,1-phenylene))dimethanol (0.009 g,
0.0266 mmol) in dichloromethane (4 mL) at 0 °C (Ar atmosphere)
then stirred at 25 °C for 1 h. The resulting solution was diluted
with ethyl acetate (15 mL) and washed with citric acid (5%,
20 mL), sodium bicarbonate (5%, 20 mL) and brine (20 mL), dried
(MgSO4) and condensed in vacuo. The residue was added dropwise
to a solution of 1,4-diazabicyclo[2.2.2]octane (DABCO) (0.006 g,
0.2 equiv) in acetonitrile (2 mL) at 0 °C. The resulting solution
was stirred at 25 °C for 90 min then condensed in vacuo. The resi-
due was recrystallized from hexanes, methanol and ethyl acetate
to afford the title compound (0.0176 g, 92%) as a yellow solid.
Mp 133–135 °C; 1H NMR (300 MHz, CDCl3): 7.44–7.51 (m, 4H),
7.28–7.37 (m, 6H), 7.01–7.08 (m, 2H), 4.85 (s, 4H),3.58 (t, 12H,
J = 7.2 Hz), d 3.14 (t, 12H, J = 7.2 Hz), 2.81 (s, 6H); 13C NMR
(75 MHz, CDCl3): 134.90, 134.85, 133.43, 130.20, 128.38, 127.12,
HRMS C42H42N2 (TOF MS ES+), m/z (M)2+: calcd 287.1674, obsd
þ
287.1667.
2.21. 4-(2-Phenylethynyl-phenylethynyl)-benzyle ester
polyethylene glycol carboxylic acid (17)
(4-((2-Phenylethynyl)phenyl)ethynyl)phenyl)methanol (0.081 g,
0.26 mmol) was added to a solution of polyethyleneglycol (PEG)
diacid (690.8 g/mol, 0.40 g, 0.58 mmol), 1-ethyl-3-(3-dimethyl-
aminopropyl)carbodiimide (0.11 g, 0.58 mmol) and 4-dimethyl-
aminopyridine (0.01 g, 0.08 mmol) in dichloromethane (8 mL).
The solution was stirred at 25 °C for 12 h, then washed with brine
(20 mL), dried (MgSO4), and condensed in vacuo. The reside was
purified by flash chromatography (dichloromethane/methanol
(1:1) to yield the title compound (0.09 g, 32%) as a yellow oil. 1H
NMR (300 MHz, CDCl3): 7.58–7.26 (m, 13 H), 5.15 (s, 2H), 3.8–
3.38 (m, PEG, 53H), 2.7 (t, 2H, J = 6.3 Hz), 2.59 (t, 2H, J = 6.0 Hz);
13C NMR (75 MHz, CDCl3): 174.1, 171.55, 138.8, 136.4, 132.0,
131.9, 128.7, 128.6, 128.3, 127.2, 126.1, 125.9, 123.5, 121.8, 93.9,
93.4, 89.0, 88.5, 70.7, 67.0, 66.8, 66.0, 35.34, 32.9, 32.1, 29.9; HRMS
C53H72O17 (MALDI) (M+Na)+ m/z calcd 1003.90, obsd 1003.30.
þ
126.52, 93.28, 91.11, 68.86, 53.71, 46.29, 39.74; HRMS C36H40N2
(TOF MS ES+), m/z (M+H)2+: calcd 264.1627, obsd 264.1614.
2.19. N-Benzyl-N,N-dimethyl-1-(4-((2-(phenylethynyl)phenyl)-
ethynyl)phenyl)methanaminium chloride (15)
Triethylamine (21.7
lL, 3 equiv) and methanesulfonyl chloride
(12.1 L, 3 equiv) was added to a solution of 4-(2-Phenylethynyl-
l
phenylethynyl)-benzyl alcohol (0.016 g, 0.0519 mmol) in dichloro-
methane (4 mL) at 0 °C (Ar atmosphere) then stirred at 25 °C for
1 h. The resulting solution was diluted with ethyl acetate (30 mL)
and washed with citric acid (5%, 30 mL), sodium bicarbonate (5%,
30 mL) and brine (30 mL), dried (MgSO4) and condensed in vacuo
. Acetonitrile (1 mL) was added and the solution was added drop-
2.22. 4,40-(4,40-(1,2-Phenylenebis(ethyne-2,1-diyl))bis(4,1-
phenylene))bis(methylene)bis(oxy)bis(4-oxobutanoic acid) (20)
wise to a solution of N,N0-dimethylbenzylamine (7.8
lL, 1 equiv)
(4,40-(1,2-Phenylenebis(ethyne-2,1-diyl))bis(4,1-phenylene))di-
methanol (0.0165 g, 0.0488 mmol) was added to a solution of 4-
dimethylaminopyridine (0.006 g, 1 equiv) and succinic anhydride
(0.0059 g, 1.2 equiv) in dichloromethane (3 mL). The mixture was
stirred at 25 °C (Ar atmosphere) for 12 h. The solution was diluted
with dichloromethane (10 mL) and washed with aqueous sodium
carbonate (5%, 25 mL). The dichloromethane layer was removed
and the aqueous layer acidified by dropwise addition of concen-
trated hydrochloric acid. This solution was back extracted with
ethyl acetate (3 ꢀ 25 mL), washed with brine (25 mL), dried
(MgSO4), and condensed in vacuo to afford the title compound
(0.0205 g, 99%) as a white solid. Mp 126–127 °C; TLC (dichloro-
methane/methanol/aqueous ammonium hydroxide = 80:19:1): Rf
0.22; 1H NMR (500 MHz, CDCl3): d 7.53–7.60 (m, 6H), 7.29–7.35
(m, 6H), 5.17 (s, 4H), 2.64–2.75 (m, 8H); 13C NMR (75 MHz,
CD3OD): d 176.16, 173.98, 138.32, 132.82, 132.66, 129.47, 129.22,
126.95, 124.16, 94.15, 89.36, 66.85, 30.05, 29.83; HRMS C32H26O8
(TOF MS ES+), m/z (M+Na)+: calcd 561.1536, obsd 561.1525.
in acetonitrile (1.5 mL) at 25 °C. The resulting solution was stirred
at 60 °C for 4 h then condensed in vacuo. The residue was dissolved
in ethyl acetate (10 mL) and the solution was washed with brine
(10 ꢀ 10 mL). The brine washings were back extracted with dichlo-
romethane (10 ꢀ 10 mL) then the combined organic layers were
dried (MgSO4) and condensed in vacuo to afford the title com-
pound (0.020 g, 83.6%) as a white solid. Mp 112–115 °C; 1H NMR
(500 MHz, CD3OD): 7.68 (d, J = 8.5 Hz, 2H) 7.50–7.64 (m, 11H),
7.33–7.45 (m, 5H), 4.63 (s, 2H), 4.61 (s, 2H), d 2.95 (s, 6H); 13C
NMR (125 MHz, CD3OD):
d 134.74, 134.50, 133.38, 133.16,
133.12, 132.72, 132.19, 130.58, 130.10, 130.00, 129.84, 129.67,
129.12, 128.87, 127.36, 127.33, 126.44, 124.54, 94.71, 93.21,
91.48, 88.94, 70.04, 69.43, 55.65; HRMS C32H28N+ (TOF MS ES+),
m/z (M)+: calcd 426.2222, obsd 426.2210.
2.20. N,N0-(4,40-(1,2-Phenylenebis(ethyne-2,1-diyl))bis(4,1-
phenylene))bis(methylene)bis(N,N-dimethyl-1-
phenylmethanaminium) methanesulfonate (16)
2.23. 4,40-(4,40-(1,2-Phenylenebis(ethyne-2,1-diyl))bis(4,1-
phenylene))bis(methyleneoxy-polyethyelene glycol alcohol)
(21)
Triethylamine (16
l
lL, 6 equiv) and methanesulfonyl chloride
(9
L, 6 equiv) was added to a solution of (4,40-(1,2-phenyl-
enebis(ethyne-2,1-diyl))bis(4,1-phenylene))dimethanol (0.007 g,
0.02069 mmol) in dichloromethane (2 mL) at 0 °C (Ar atmosphere)
then stirred at 25 °C for 1 h. The resulting solution was diluted
with ethyl acetate (20 mL) and washed with citric acid (5%,
30 mL), sodium bicarbonate (5%, 30 mL) and brine (30 mL), dried
(MgSO4) and condensed in vacuo. This residue was diluted with
acetonitrile (1 mL) and added dropwise to a solution of N,N0-dim-
4,40-(4,40-(1,2-Phenylenebis(ethyne-1,2-diyl))bis(4,1-phenylene))-
bis(methylene)bis(oxy) bis(4-oxobutanoic acid) (0.004 g, 0.00742
mmol) was added to a solution of 1-ethyl-3-(30-dimethylaminopro-
pyl)carbodiimide (EDCI) (0.0031 g, 2.2 equiv) and 4-dimethylamino-
pyridine (DMAP) (0.0020 g, 2.2 equiv) in dichloromethane (1.5 mL)
at 25 °C (Ar atmosphere). The mixture was refluxed for 4 h then
ethylbenzylamine (6.1
l
L, 2 equiv) in acetonitrile (1.5 mL) at 0 °C.
cooled to 25 °C and 400Mn polyethylene glycol (5.8
lL, 2.2 equiv)
The resulting solution was stirred at 60 °C for 3 h then condensed
was added. The solution was stirred for 48 h at 25 °C, then diluted

6300
P. LaBeaume et al. / Bioorg. Med. Chem. 17 (2009) 6292–6300
with dichloromethane (10 mL), washed with brine (2 ꢀ 10 mL), dried
(MgSO4), and concentrated in vacuo. The crude residue was purified
via preparative thin layer chromatography (90:9:1 CH2Cl2/MeOH/aq
NH4OH) to yield the title compound (3.7 mg, 39%) as a yellow oil.
TLC (dichloromethane/methanol/aqueous ammonium hydrox-
ide = 80:19:1): Rf 0.31; HRMS C66H90O24 (MALDI): m/z (M+2Na)2+
calcd1285.43982, obsd 1287.44434.
the mixture centrifuged for an additional 60 min. The concentrate
(0.7 mL) was collected and analyzed by UV–vis scan in the 200–
600 nm range.
2.27. 4-(2-Phenylethynyl-phenylethynyl)-benzyl thiol gold
nanoparticle conjugate (27)
A solution of gold(III) chloride trihydrate (0.100 g, 0.25 mmol)
in water (10 mL) was stirred for 10 min. To this was added solution
of tetraoctylammonium bromide (0.5 g, 0.91 mmol) in toluene
(30 mL) and the combined solution was stirred for 30 min. (4-
2.24. Enediyne antibody conjugate (22)
A solution of the 2G4 antibody (0.0075 g, 1.5 mL water), 1-
ethyl-3-(3-dimethylaminopropyl)carbodiimide (0.040 g, 0.21 mmol)
and S-NHS (0.045 g, 0.21 mmol) was added to a solution of 21 in
phosphate buffered saline (0.5 mL, pH 7.4).
The mixture was stirred for 5 h at 25 °C then purified via dialy-
sis using a spectrum dialysis bag (250 kDa) against distilled water
and stirred at 25 °C for 12 h. The mixture was kept at ꢁ80 °C for 2 h
followed by freeze-drying (under vacuum at ꢁ48 °C for 24 h) to
solidify the purified title compound as a white solid (0.020 g,
70%). ELISA testing was performed using unconjugated 2G4 anti-
body as a control, using absorbance profile to confirm conjugation
of the antibody to the PEG-ylated enediyne.
((2-(Phenylethynyl)phenyl)ethynyl)phenyl)methanethiol
(0.1 g,
0.31 mmol) was added and the mixture stirred for an additional
20 min. An aqueous solution of sodium borohydride (0.126 g,
3.33 mmol, 0.37 M) was added dropwise. The solution was stirred
for 2 h at 25 °C then the organic phase was washed with water
(3 ꢀ 20 mL) and concentrated in vacuo to approx. 5 mL. The solu-
tion was diluted with ethanol (25 mL) and allowed to stand over-
night at ꢁ20 °C. The precipitated nanoparticles were collected by
vacuum filtration and washed sequentially with water (5 ꢀ
20 mL), ethanol (5 ꢀ 20 mL), acetonitrile (5 ꢀ 20 mL), and acetone
(5 ꢀ 20 mL), to yield the title compound (0.1 g) as a black powder.
The gold nanoparticles were characterized by high resolution
transmission electron microscopy (TEM). TEM samples were pre-
pared by spreading one drop of a 1 mg/mL clustered toluene solu-
tion onto a standard carbon-coated Formvar film on a copper grid
(200 mesh). Average core diameter was found to be 5.0 nm.
2.25. N-(3,3-Dimethoxy-propoxy)-PEG 4-(2-phenylethynyl-
phenyletyhyl)-phenyl acetamide (23)
N,N0-Dicyclohexylcarbodiimide (0.019 mg, 0.090 mmol) was
added to a solution of 4-(2-phenyletheynyl-phenylethynyl)-phenyl
acetic acid (0.033 g, 0.090 mmol) in dichloromethane (15 mL). The
solution was stirred for 10 min then 400Mn PEG amine (0.048 g,
0.09 mmol) was added and the solution was stirred for 12 h at
25 °C. The solution was concentrated in vacuo and the residue
was subjected to column chromatography (alumina, 100% acetone)
to yield the title compound (0.0668 g, 96%) as an oil. 1H NMR
(300 MHz, CDCl3): 7.56–7.20 (m, 13H), 4.51 (t, J = 6.3 Hz, 1H),
3.67–3.38 (m, 16 ꢀ CH2), 3.33 (s, 6H), 1.88 (q, J = 6.3 Hz, 2H); 13C
NMR (75 MHz, CDCl3): 170.68, 135.81, 132.24, 132.05, 132.01,
131.85, 129.64, 128.24, 126.03, 125.96, 123.48, 122.36, 102.44,
93.81, 93.55, 88.78, 88.49, 70.75, 70.50, 69.92, 67.39, 53.27,
43.78, 39.67, 33.23; HRMS C43H55NO10 (ESI), m/z (M+Na)+: calcd
768.3721, obsd 768.2378.
Acknowledgements
We thank the NIH (RO1GM57123) for financial support of this
work, and Amit Kale and Dmitriy Mongayt for performing ELISA
assays.
References and notes
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2.26. Enediyne antibody conjugate (24)
Deprotection of the 23 was achieved by treatment of an ace-
tone/water (1:1) solution of the propionaldehyde dimethylacetal
O-PEG derivative (0.20 g, 0.13 mmol) with Amberlyst 15 ion-ex-
change resin (1.24 g). The reaction mixture was stirred for 24 h
at 25 °C. The mixture was filtered over a glass plug and concen-
trated in vacuo. The crude oil was crystallized from a 1:1 dichloro-
methane/ether mixture (ꢁ78 °C) to yield N-(3-acetaldehyde)-PEG
4-(2-phenylethynyl-phenyletyhyl)-phenyl acetamide (0.208 g,
100%) as clear oil; 1H NMR (300 MHz, CDCl3): 9.78 (s, 1H), 7.75–
7.20 (m, 13 H), 4.0–3.54 (m, 26H), 3.48–3.38 (m, 4H), 2.71–2.61
(m, 2H); HRMS C41H49NO9 (ESI), m/z (M+CH3OH+H)+: calcd
732.3232, obsd 732.4016.
HEPES buffer (500 lL) were added to a solution of 2G4 (0.001 g,
0.395 mL) and N-(3-acetaldehyde)-PEG 4-(2-phenylethynyl-phen-
yletyhyl)-phenyl acetamide (0.002136 g, 0.00265 mmol) in
dimethylformamide (100 lL). Sodium cyanoborohydride (2.513 g,
40 mmol) was added and the mixture was stirred at 4 °C for
14 days. The mixture was transferred to the MACROSEP 50 K OME-
GA (PALL Life Science) resin with phosphate buffered saline (1 mL,
pH 7.4) and then centrifuged for 30 min (5000 g, ꢁ4 °C). An addi-
tional portion of phosphate buffered saline (1 mL) was added and